Methods for volumetric determination of fresh endocrine glands.

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blETHODS F O R VOLUMETRIC DETERMINATION O F
F R E S H ENDOCRINE G L S N D S
C . A . SWINYARD
Xedical Colle,ye of tRe State of South Carolina, Charleston
TN TRODU CTI ON
Many papers have been published concerning the volume
of endocrine glands under normal, experimental and pathological conditions. A study of the literature indicates that
a number of methods have been used f o r the determination of
the volume of glands. Most of the volumetric measurements
however, have been made by projecting serial sections of the
glands on paper and either measuring the area of the sections
with a planimeter or by using the paper weight method suggested by Hammar ( ’14). Both of these methods are time
consuming and expensive and even though a correction is made
for shrinkage a number of other sources of error are present
which may affect the final volume to a considerable degree.
F o r these reasons it appears to be desirable to call attention
to three inexpensive, rapid and accurate methods of volumetric
determination. One of the methods to be discussed is new
while the other two have apparently been neglected.
METHODS
Available methods for ascertaining the volume of fresh
glands fall into three general categories :
1. The simple water displacement method in which the
amount of water displaced by the gland may be measured by
volume or bj7 weight.
I \\ish to express iny sincere appreciation to Dr. R. E. Seaminoii of the
University of Minnesota for constructive criticism and eouasel. Thanks are also
clue the members of the department of biochemistry of this institution f o r heIpful
siiggestions and for placing a Christian Beeker Chainomatic analytical balance
a t m y disposal.
71
I l l E \NATO\ITCAI
R > C f l R D , \OL
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72
C . A. SWINYABD
2. The water displacement method based on Archimedes
principle in which the volume is obtained by subtracting the
weight of the gland in water from its weight in air.
3. The method whereby the volume may be obtained when
the weight of the gland in air and the specific gravity of the
tissue are known.
The simplest method of obtaining the volume of a fresli
gland is to place the tissue in a known quantity of water in
a graduated cylinder and determine the amount of water
displac,ed. This procedure is reasonably exact for a large
gland but the writer has found it very difficult to measure
accurately the amount of displaced water of a small gland
such as the thyroid of a rat. I n an attempt to obtain the
volume of r a t thyroids with this procedure the smallest
accurately graduated cylinder of suitable caliber offered great
difficulty in correctly reading the change in the meniscus.
When using this method, the weights when divided by the
volume obtained resulted frequently in a specific gravity less
than unity yet the glands would sink rapidly ~7he11placed in
distilled water. However, Scammon ( '38) has improved the
water displacement procedure by developing a micro-method
of measuring change in the meniscus of a column of fluid which
is accurate to within 0.004 cc.
Another method of measuring the displaced water is by
weight. This is most readily accomplished with a ppcnometer.
Carlson ( ' 3 7 ) used this method to obtain the voliinic of the
suprarenal glands of white mice. I n this procedure the gland
is first weighed in air and then a pycnometer is filled with
distilled water and weighed. Finally the gland is placed in
the pycnometer which is then filled with water and weighed.
The volume of the gland is then equal to the weight of the
gland in air plus the weight of the pycnometer filled with
distilled water minus the weight of the ppcnometer containing
the gland and water. The writer has found this method to
require the utmost care in order to obtain accurate results.
The capillary tube of the pycnometer must be very small.
All traces of moisture should be removed from the outside
Y O L U M E O F E N D O C R I N E GLANDS
73
of the bottle and in addition a temperature correction must
be applied. I n my experience this procedure proved t o be
less accurate than the two following methods.
The method based on Archimedes principle has been used
snccessfullp by Stoeltzner ('06) to sliow the effect of fixation
011 the volume of an organ. If it i s undcsirahlc t o place tlie
fresh tissue in water, normal saline map be used and the
volume computed by means of the following formula as used
hy Stoeltzner :
x - (P - cl) x 100
I;
Where :
X = Volume of the gland
p =Weight of the gland in air
a =Weight of the gland i n ph~siologicalsaliiir
R = Wciglit of 100 cc. of physiological saline solutioii
This method also requires the greatest care hecanse a slight
error in the weight of the gland in water will result in considerable error in volume. F o r example, in a 25 mg. r a t
thyroid an error of only 0.5 me. in tlie weicht in water will
introduce an error of 2.1176 in the volume. The water offers
some resistance to the movement of the gland when weighing
and for this reason it is recommended that the numerical figure
used as the weight in water should he an average taken from
at least five weight measurements. The weights of the glands
can be obtained in a very short time if the balance is equipped
with a magnetic damper.
Although the density of the water in which the glands are
weighed varies with the temperature, the error introduced
by this variable is negligible. By assuming the density of
water to be unity and makiny correction f o r the absolute
density a t the highest observed temperature an error of only
0.28% was introduced in the final volume.
I n the third volumetric method to be discussed the gland
is carefully weighed in air, then the specific gravity determined
and the volume obtained hy dividing the weight by the specific
gravity. The specific gravity is determined by a modification
of the method which Hammerschlag (1892) devised for the
74
C. A. SWINYARD
determination of the specific gravity of blood. As far as the
writer can ascertain this procedure has never been adapted
for use in volume determination of glands. The method consists of placing the gland in the center of a solution of glycerol
and water of such density that the gland neither sinks nor
rises to the surface. When such a condition prevails it is
assumed that the density of the Auid and the gland is the
same and the specific gravity of the fluid is then obtained with
a hydrometer or pycnometer. I n this study the specific gravity
of the glycerol-water mixture was determined by means of
a precision hydrometer. The accuracy of the hydrometer was
checlied by determining the density of the same fluid first, by
means of the hydrometer then by use of a pycnometer. The
average per cent difference in the specific gravity in ten
trials amounted to 0.20%. An error of this magnitude results
in a volume difference of only 0.04 cu.mm. in a 25 cu.mm. rat
thyroid and was not considered to be significant.
Temperature variations also need not he considered with
the third method. Bosart ('27) has shown that the true
specific gravity of a 20% aqueous glycerol solution at 15°C.
is 1.04930 while the same solution at 25°C. has a specific
gravity of 1.04830. Therefore a change of 10°C. (18°F.)
brings about a n error i n final volume of only 0.10%. I n view
of this small difference i t is believed that the density of water
can be assumed to be unity and ordinary temperature variations are, therefore, unimportant. The hydrometer xsed in
this investigation was calibrated at 60°F. and the scale
graded in 0.002 intervals. A uriiiometer has a suitable range
for most glands and is graduated at 0.001 intervals. I n a
test a urinometer was found to be satisfactory when checked
with a pyciiometer and a hydrometer which was accompanied
with a bureau of standards certificate. A correction factor
can be applied to the urinometer readings.
I n the routine use of this method a series of test tubes are
filled with glycerol-water mixtures of such proportion that
the difference iii specific gravity hetween adjacent tubes is
0.002. The gland is placed on a mire spoon and lowered to
V O L U M E O F ENDOCRINE GLANDS
75
the center of the column of fluid. The spoon is quickly lowered
to the bottom of the tube. If the gland sinks or rises to the
surface it is removed, the excess fluid is blotted off and the
gland then placed in a more appropriate tube. The specific
gravity of the fluid in which the gland neither sinks nor rises
to the surface is then measured with a pycnometer or a
hydrometer of known accuracy.
DISCIWSION
The accuracy of the method based on Archimedes principle
and the weight-specific gravity method mas tested by applying
both procedures on a series of ten fiber cylinders and twenty
human, dog and rat endocrine glands. The cylinders were
machined to 0.002 of an inch and the true volume of each
cylinder was obtained arithmetically ( nr2h) from measurements made with a micrometer. The volume of the cylinders
ranged from 1579.9 cu.mm. t o 13.1 cu.mm. and mas used as a
t m c volume with which the volumes obtained by the abovementioned methods were compared. The results are shown in
table 1.
The greatest difference between the triie volume and the
observed volume was 1.63% with an average of 0.54% difference. I n seven of the ten cylinders the standard error
of the greatest difference in volume was of no statistical
significance. I n 70% of the cylinders the volume obtained
b;v the weight-specific gravity method was closer t o the true
volume than that obtained by Archimedes principle. The
specific gravity of the cylinders obtained by glycerol-water
suspension averages within 1% of the specific gravity obtained by dividing the weight by the arithmetic volume. I n
the cylinders in which the high specific gravity required a concentrated glycerol solution or extended beyond the glycerol
range the specific gravity was checked by suspension in a
chloroform-ether mixture. I n 90% of the cylinders the
standard deviation, probable error and coefficient of variation was smaller in the weight-specific gravity method than
Q,
-a
TABLE 1
1
volume diff ereiice in cubic millimeters
20.3203
Maximum volume diff ereiice
0.40
C'oefficient of variation in per cent
0.018
Weight in air in milligrams
1961.3
Weight in water in milligrams
387.66
Volume in cubic millimeters froiii
Archimedes principle
1573.6
Probable error in cubic millimeters 50.3853
Standard deviation in cubic millit0.5712
meters
CYLINDER NUMBER
11.505
7.646
15.298
7.622
11.304
11.457
7.517
7.637
5
20.3137 k0.4222
0.091
0.271
425.1
198.3
80.64
43.2
155.77
t0.2847
7.633
2.097
1.229
1.229
k0.3674 f0.2629
0.11
0.18
0.15
0.09
20.2867 k0.1662
k0.1095
0.031
20.2898
0.731
49.7
10.2
39.60
k0.1954
7.641
2.668
8
1.263l
0.726
20.2870
3Z0.1459
0.64
k0.1081 20.0340
0.086
0.103
39.35
104.57
k 0 . 0 7 2 9 20.0229
1.275'
20.1939
0.186
104.12
39.49
k0.1307 "0.1935
20.1862
0.179
133.3
29.2
103.99
20.1255
5.090
5.100
7
k 0 . 2 1 9 1 C0.299:)
0.42
0.36
20.1568
0.100
&0.2464
0.046
310.4303 50.2826
0.040
0.036
1.273'
20.1166
0.075
155.32
t0.1037
1.235
-+0.3610
0.104
697.70
528.29
344.04
165.9
t0.2902 20.1906 f 0 . 1 6 6 1 k0.0738
1.064
k0.4317
0.081
20.4857 e0.3498
0.050
0.041
163.5
527.36
697.44
344.46
155.12
k 0 . 3 2 7 t 20.2359 -1-0.2911 k 0 2434 ?0.07 86
k0.5509 20.3921 k0.3769
0.056
0.047
0.071
837.9
241.5
649.5
160.46
78.04
121.94
698.14
165.5
344.34
528.35
k0.3715 k0.2644 20.2542 kO.2115
4
3
2
20.0313
0.236
13.21
10.0211
1.262'
50.1166
0.875
13.32
50.0786
0.468
16.6
3.3
?O.O(ilfj
13.16
k0.0415
2.633
2.523
1-63
1.21
3Z0.0982 t 0 . 0 6 5 7
20.1058
0.403
26.20
k0.0713
1.261'
k0.1356
0.520
26.06
20.0914
k0.1421
0.536
33.0
7.0
26.49
k0.0958
5.101
2.571
10
Comparison of t h e uolzime of ten macltined fiber cylindem as obta n e d b y a r z t h i n e t i c ?nemure, Arclizinedes p ~ i n c f p ' eund by specific
qravzty. A11 figuies r e p r e s e n t an aceruge o f fire meusicrements
77
VOLUME OF ENDOCRINE GLANDS
in the other method. These figures indicate the greater uniformity and accuracy of the weight-specific gravity method.
The application of these two procedures to the endocrine
glands is shown in table 2. The volume of the glands ranged
from 5740.8 cu.mm. t o 18.3 cu.mm. The maximum difference
in volume was 1.8% in the case of one rat thyroid gland.
TABLE 2
Comparison of t h e volume of eitdacrine glands as determined b y Archimedes
principle and weight-specifio gravity method
GLAND
WEIGHT
I N AIR
N GRAMS
WEIGHT
N XXTER
N GRAMS
VOLUMdl
N OUBIC
OENTIMETERS
jPECIFIC
PRAVITY
Human suprarenal
Hnnian suprarenal
Human suprarenal
Human suprarenal
Dog thyroid
Dog thyroid
Dog thyroid
Dog thyroid
Dog suprarenal
Dog suprarenal
Dog suprarenal
Dog suprarenal
Rat suprareiial
Rat suprareiial
Rat suprarenal
Rat suprarenal
Rat thyroid
Rat thyroid
Rat thyroid
Rat thyroid
4.1916
5.9237
3.8 7 57
3.8923
1.4056
0.5894
1.0307
0.9386
0.8699
0.8473
0.5521
0.8693
0.0421
0.0330
0.0348
0.0329
0.0281
0.0241
0.0230
0.0191
0.1460
0.1829
0.1597
0.1466
0.0904
0.0247
0.0600
0.0567
0.0344
0.0348
0.0205
0.0354
0.0024
0.0013
0.0011
0.0016
0.001.5
0.0013
0.0013
0.0008
4.0456
5.7408
3.7160
3.7457
1.3152
0.5647
0.9707
0.8819
0.8355
0.8125
0.5316
0.8339
0.0397
0.0317
0.0337
0.0313
0.0266
0.0228
0.0217
0.0183
1.038
1.036
1.041
1.041
1.068
1.039
1.053
1.057
1.040
1.040
1.037
1.038
1.043
1.039
1.035
1.039
1.046
1.052
1.038
1.038
VOLUME I N
CUBIO
CENTIMETERS
4.0381
5.7178
3.7230
3.7390
1.3161
0.5672
0.9788
0.8879
0.8364
0.8147
0.5324
0.8374
0.0403
0.0317
0.0336
0.0316
0.0268
0.0229
0.0221
0.0164
W R CENT
FFERENCE
N VOLUME
0.19
0.41
0.19
0.18
0.07
0.45
0.83
0.68
0.11
0.28
0.16
0.42
1.50
0.00
0.30
1.00
0.80
0.50
1.80
0.55
The average difference was 0.52%. This close agreement
is believed to be indicative of the reliability of the met,hods.
The weight-specific gravity method is more accurate and is
easier to handle. These procedures do not interfere with
subsequent histological study. Although the section method
must be used when the volume of a lobe or the medulla of a
gland is desired, the weight-specific gravity volume can be
78
C. A. SWINYARD
quickly obt,aiiied a n d serve as a check for true volume or
shrinkage correction.
CONCLUSIONS
1. The commonly used projection method for volumetric
determination of glands is time consuming and expensive.
2. The water displacement method is inaccurate with small
glands.
3. The volume of a gland can he determined within 2% either
by use of Archimedes principle o r by computing volume from
weight and specific gravity.
4. The specific gravity of a gland can be determined within
1 % by suspending the gland i n a glycerol-water mixture and
determining the specific gravity of the fluid with a p p i o m e t e r
or a hydrometer of known accuracy.
5. The volume of a gland can be most accurately determined
by the -\T.eiglit-specificgravi t p method.
LITERATURE CITED
EOSART,
L. W., AND A. 0. SNODDY1927 New glycerol tables-tables f o r specific
gravity and per eelit of glycerol-thermal
expansion of aqueous soliitioiis in terms of specific gravity. J. Ind. and Eug. Chemistry, 1701. 19,
pp. 506-510.
CARLSON,
H., E. GUSTAFSSON AND K. L. MOLLER 1937 Quantitative uiikromorphologische Studieii uber die Nebeiiiiiereii einjahriger weisser Miiuse
uiiter besoiiderer Beriicksichtigung voii Geschlechtsverschiedeuheiten.
Srparat ur Upsala Lakareforeiiiiigs f orhaiidlinger. Ny foljd, Ed.
XLIII, S. 49-62.
I I A i i h f m , J. A. 1914 hlethode, die Meiige der Rinde und des Marks der Thymus,
Sonic die Anzahl mid die Grosse der Hassalsclicii Korper zahlenmiiqsig
festuzustellen. Zeitschr. f. aiigcwaiidte h a t . ti. I(oiistitutioiislehre,
Rd. 1, S. 312-396.
HAXNEESCHLAG,
A. 1692 Ein iieue Methode zur Bestimmuiig des speeifisclien
Gewichts des Blutes. Zeitschr. f. Klin. Med., Ed. 20, S 444-456.
SCAMMON,
R.E. 1938 Personal communication.
STOELTZNER,
HELEN 1906 Der Einfluss der Fixierung auf das Volumeu der
Orgaiie. Ztschr. f. Wissenscliaftliche Mikroskopie und f u r hlikroskopisclie Technick, Rd. XXITI, S. 14-25.